Isoforms of starch branching enzyme II (SBE-IIa and SBE-IIb) from wheat

ABSTRACT

A class of wheat SBEII genes, called SBEII-1, can be used to influence properties of starch produced by a plant, including the gelatinization temperature of starch. Such starch is useful, for example, in certain industrial applications for the preparation or processing of foodstuffs such as bakery products. One aspect of the present invention provides a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:7.

This application is a divisional of U.S. patent application Ser. No.10/818,770 filed Apr. 6, 2004, now U.S. Pat No. 7,217,857 which is adivisional of U.S. patent application Ser. No. 09/786,480 filed Sep. 17,2001, now U.S. Pat. No. 6,730,825 which is a §371 national stage filingof PCT/GB99/03011, filed Sep. 9, 1999 (published in English on Mar. 23,2000 as WO 00/15810) and claiming priority to EP 98307337.0 filed Sep.10, 1998, all of which applications are incorporated herein by referencein their entirety.

FIELD OF THE INVENTION

This invention relates generally to plant starch compositions, andconcerns novel nucleotide sequences; polypeptides encoded thereby;vectors and host cells and host organisms comprising one or more of thenovel sequences; a method of altering one or more characteristics of aplant; a plant having altered characteristics; starch obtained from suchplants; and uses of the starch.

BACKGROUND TO THE INVENTION

The majority of developments in cereal science in the recent past haveconcentrated primarily on the functionality of the gluten proteinsub-units and their role in bakery systems. This has been greatlyfacilitated by the abundance of natural variation between cultivatorsfor the gluten protein sub-unit components.

In contrast, although flour from commercially grown wheat varietiescontains approximately 75-85% starch, the role of starch from a breedingperspective has been overlooked; this is largely due to the difficultyof measuring differences in starch structure. Of the limited amount ofwork that has been carried out however, there appears to be a lack ofnatural variation between different wheat cultivars. With the advent ofrecombinant DNA and gene transfer technologies it is now possible tocreate new variation in planta, therefore directly modifying starchcomposition in wheat becomes a realistic target.

Starch is the major form of carbon reserve in plants, constituting 50%or more of the dry weight of many storage organs, e.g. tubers, seeds ofcereals. Starch is used in numerous food and industrial applications. Inmany cases, however, it is necessary to modify the native starches, viachemical or physical means, in order to produce distinct properties tosuit particular applications. It would be highly desirable to be able toproduce starches with the required properties directly in the plant,thereby removing the need for additional modification. To achieve thisvia genetic engineering requires knowledge of the metabolic pathway ofstarch biosynthesis. This includes characterisation of genes and encodedgene products which catalyse the synthesis of starch. Knowledge aboutthe regulation of starch biosynthesis raises the possibility of“re-programming” biosynthetic pathways to create starches with novelproperties that could have new commercial applications.

The most significant property of starch derives from the ability of thenative granular form to lose its order and to swell and absorb waterupon suitable treatment, thereby conferring viscosity and texture, in aprocess known as gelatinisation. Gelatinisation has been defined (W AAtwell et al, 1988) as “. . . the collapse (disruption) of molecularorders within the starch granule manifested in irreversible changes inproperties such as granular swelling, native crystallite melting, lossof birefringence, and starch solubilisation. The point of initialgelatinisation and the range over which it occurs is governed by starchconcentration, method of observation, granule type, and heterogeneitieswithin the granule population under observation”.

14 molecules of water per molecule of anhydrous glucose, i.e. a minimumof 75% water, are necessary for full starch gelatinisation (Donovan,1979). Starch gelatinisation is usually, caused by heat, but can becaused by physical damage and some chaotropic agents, mainlydimethylsulphoxide (DMSO), urea, calcium chloride, strong base and acid.

The various events taking place during gelatinisation can be followed byvarious methods, including birefringence, X-ray diffraction,differential scanning calorimetry (DSC), ¹³C NMR. Swelling can bemonitored by various methods, particularly rheology.

Differential scanning calorimetry (DSC) is a destructive method whichrecords an endothermic event on heating of granules, generally thoughtto measure the temperature and the endothermic energy (delta H) requiredfor the melting of the native crystallites. Starch gelatinisationtemperature is independent of water content above 75% water (describedas excess water), but increases when water is limited (Donovan, 1979).

The rate and extent of starch granule swelling upon heating dictate thetype of viscosity development of aqueous starch suspensions on heating.Swelling behaviour is therefore of utmost technological importance.Viscosity increase on heating can be conveniently measured by aBrabender amylograph (Brabender is a Trade Mark) (Kennedy and Cabalda,1991) or using a Rapid Visco analyser (Rapid Visco is a Trade Mark fromNewport Scientific, Australia). FIG. 1 is a typical viscoamylgraphprofile for wheat starch, produced in this way, showing changes instarch during and after cooking. As starch granules swell on uptake ofwater, in a process known as pasting, their phase volume increases,causing an increase in viscosity. The onset of pasting is indicated at Ain FIG. 1. Peak viscosity, indicated at B in FIG. 1, is achieved whenmaximum phase volume is reached. Shear will then disrupt/causefragmentation of the swollen granules, causing the viscosity todecrease. Complete dispersion is indicated at C in FIG. 1. This has beenconfirmed by an oscillatory rheology study of starch pastes at variousstages of the viscosity profile (Svegmark and Hermansson, 1990). Theviscosity onset temperature and peak viscosity are indicative of theinitiation and extent of swelling, respectively. On cooling, leachedamylose forms a network in a process involving reassociation ofmolecules, or retrogradation, causing an increase in viscosity asindicated at D in FIG. 1. Retrogradation (or set-back) viscosity istherefore indicative of the amount of amylose leached out of thegranules.

The properties of wheat starch are useful in a large number ofapplications and also non-food (paper, textiles, adhesives etc.)applications. However, for many applications, properties are not optimumand various chemical and physical modifications well known in the artare undertaken in order to improve useful properties. Two types ofproperty manipulation which would be of use are: the controlledalteration of gelatinisation and pasting temperatures; and starcheswhich suffer less granular fragmentation during pasting thanconventional starches.

Currently the only ways of manipulating the gelatinisation and pastingtemperatures of starch are by the inclusion of additives such as sugars,polyhydroxy compounds of salts or by extensive physical or chemicalpre-treatments. The reduction of granule fragmentation during pastingcan be achieved either by extensive physical pre-treatments or bychemical cross-linking. Such processes are inconvenient and inefficient.It is therefore desirable to obtain plants which produce starch whichintrinsically possesses such advantageous properties.

Starch consists of two main glucose polysaccharides: amylose andamylopectin. Amylose is a generally linear polymer comprising α-1,4linked glucose units, while amylopectin is a highly branched polymerconsisting of an α-1,4 linked glucan backbone with α-1,6 linked glucanbranches. In wheat endosperm amylopectin constitutes approximately 70%of the total starch content, with the balance being amylose. Amylopectinis synthesised through the concerted action of several enzymes,including soluble starch synthase(s) (SSS), starch branching enzyme(s)(SBE), starch de-branching enzyme(s) (DBE). The physical properties ofstarch are strongly affected by the relative abundance of amylose andamylopectin, therefore SSSs, SBEs and DBEs play a key role indetermining both starch quantity and quality. As such, one approach tomanipulating starch structure would be to modify the expression of theenzymes involved in starch biosynthesis in the endosperm using atransgenic approach.

SBE catalyses the formation of the α-1,6 linkages, creating branchpoints in the growing starch molecule, via hydrolysis of an α-1,4linkage followed by reattachment of the released α-1,4-glucan chain tothe same or another glucosyl chain. This reaction also provides a newnon-reducing end for further elongation of the original α-1,4-glucanchain.

Multiple isoforms of starch branching enzyme have been described,biochemically, from a number of species including maize (Boyer andPreiss, 1978), rice (Nakamura et al., 1992), pea (Smith, 1988), potato(Khoshnoodi et al., 1993) and wheat (Morell et al., 1997). Morerecently, genomic and cDNA sequences for SBE have been characterisedfrom several species including maize (Baba et al., 1991; Fisher et al.,1993; Gao et al. 1997) pea (Burton et al., 1995), potato (Kossmann etal., 1991), rice (Nakamura and Yamanouchi, 1992; Mizuno et al., 1993),Arabidopsis (Fisher et al., 1996), cassava (Salehuzzaman et al., 1992),and wheat (Rapellin et al., 1997, Nair et al., 1997, Rahman et al.,1997). Sequence alignment of these SBEs revealed a high degree ofsequence conservation at the amino acid level and that the SBEs may begrouped into two distinct families, generally known as SBEI and SBEII.Further, analysis indicates that within a species there is generally ofthe order of 50% homology between the two families, SBEI and SBEII,while there is often greater homology within the two families betweenspecies.

Maize is unusual in that the maize SBEII family is thought to comprisetwo different members, known as SBEIIa and SBEIIb. There has beencontroversy over whether the SBEIIa and IIb enzymes are in fact a)encoded by genes at two different loci, and b) whether the genesrepresent different alleles at a single locus. Fisher et al (1996) andGao et al (1997) have provided evidence that SBEIIa and SBEIIb areencoded by independent genes. However, there is no conclusive evidencethat both isoforms exist together in any one maize genotype. The DNAclones for the two published gene sequences were purified from differentgenotypes of maize and it is thus possible that they represent differentalleles of a single locus. In summary, in maize, three distinct SBEgenes have been characterised to date (Baba et al., 1991; Fisher et al.,1993; Gao et al., 1997). SBEI is distinct from SBEIIa and SBEIIb inamino acid composition, substrate specificity, kinetic properties, andimmunological reactivities, whereas SBEIIa and SBEIIb are similar inthese respects (Guan and Preiss, 1993; Preiss 1991; Takeda et al.,1993). At the amino acid level the sequence exhibits approximately 50%homology with the SBEIIa and SBEIIb sequences, whereas SBEIIa and SBEIIbexhibit approximately 80% homology to each other.

Prior to the present invention, maize was unique in having SBEIIa- andSBEIIb-type enzymes. Although Arabidopsis has two SBEII family members,the sub-division in Arabidopsis does not appear to conform to that seenin maize: the Arabidopsis sub-family members do not obviously fall intothe IIa and IIb categories as do the maize sequences. Both of theArabidopsis SBEII genes have similar levels of homology to both themaize SBEII genes, SBEIIa and SBEIIb, but the similarities are notsufficient to be able to place the Arabidopsis genes into the sameSBEIIa and SBEIIb categories as for maize. Indeed, the data, ifanything, suggests that the Arabidopsis SBEII genes do not fall into themaize IIa and IIb categories. For barley, two forms of SBEII had beenpartly characterised. Although these have been called SBEIIa and SBEIIb,only a very limited amount of sequence information had been published(Sun et al, 1995) and it was not possible to infer or conclude thatthese forms correspond to the IIa and IIb categories of maize. In fact,based on the available barley sequence information both of the barleySBEII sequences (SBEIIa and SBEIIb) would appear to show greaterhomology to maize SBEIIa than to maize SBEIIb.

For all other plant species for which SBEII sequences have beenidentified and published, including potato, pea, rice, cassava, wheatand barley, no sub-division of the SBEII family comparable to the SBEIIaand SBEIIb division of maize has been made.

Studies of purified SBEI and SBEII demonstrate that these isoformsdiffer in their specificity for a substrate with respect to both chainlength and degree of branching. In maize, SBEI and SBEII show distinctbranching activities in vitro, with SBEI showing a higher rate ofbranching of an amylose substrate when compared to SBEII whereas bothSBEIIa and IIb show higher rates of branching than SBEI when acting uponan amylopectin substrate (Guan and Preiss, 1993). Furthermore, maizeSBEI preferentially transfers longer glucan chains (average chainlength=24) than SBEII (average chain length=21(IIa) and 22(IIb)) (Takedaet al., 1993). A similar observation has been reported for SBEI andSBEII isoforms from wheat and pea (Morell et al., 1997; Smith, 1988).Mutational studies in maize, rice and pea demonstrate that high amylosemutants in each case are deficient in the branching enzyme activityanalogous to maize SBEII (Martin and Smith, 1995; Morell et al., 1995).However, the linkage between the biochemical observations and thegenetic evidence suggesting the differences in the roles remainsunclear.

The present invention is based on the unexpected discovery of a novelclass of SBEII genes in wheat, referred to herein as SBEII-1. The novelSBEII-1 gene sequence has strong homology with the maize SBEIIb gene.The wheat SBEII-1 genes are thought to be functionally equivalent to themaize SBEIIb gene, and on this basis it is believed that manipulation ofthe wheat SBEII-1 gene is likely to influence starch propertiesincluding starch gelatinisation temperature, in a manner analogous tomanipulation of the maize SBEIIb gene as described in WO 97/22703.

In summary, although two different SBEII gene sequences are known frommaize, Arabidopsis and barley, as discussed above, prior to the presentinvention there was no reason to expect that wheat would show a similarsub-division of SBEII genes as is seen for maize. The two ArabidopsisSBEII genes show a different sub-division, and prior to the presentinvention there was insufficient evidence to determine whether the twobarley SBEII sequences belonged to the maize-type sub-division. That is,prior to the present invention there was no reason to expect that wheatwould have two similar SBEII members comparable to those of maize.Subsequent to the present invention Sun et al (1998) have presented datawhich indicates that the barley sequences do indeed sub-divide in asimilar manner to the maize SBEIIa and IIb sequences and the wheatSBEII-2 and SBEII-1 sequences discussed in this document.

The present inventors have used the high degree of sequence conservationbetween several SBE gene sequences to design oligonucleotide primers tomotifs which are specific to either SBEI or SBEII families and have usedthese primers to amplify cDNA sequences from developing endosperm ofwheat.

When this work was started, a single partial length wheat SBE cDNA clonehad been reported (Mousley, 1994). Multiple sequence alignment of thiswheat SBE sequence with other published SBE sequences from a number ofplant species revealed a number of motifs which were highly conserved.Oligonucleotide primers designed to be complementary to these motifswere used to clone 3′ partial length cDNA clones of wheat SBEII.Alignment of the cDNA clone sequences indicated that the clones could bedivided into two classes, which the inventors have designated SBEII-1and SBEII-2, which showed greater than 90% similarity to members withina class but only 60% similarity between classes. Significantly,comparison between representative sequences from each class withpreviously identified wheat SBEII clones, pWBE6 (Mousley, 1994) andSBEII (Nair et al., 1997), showed that each appear to be homologues ofthe SBEII-2 class. The cloning of a wheat SBEII-1 cDNA is novel.

SUMMARY OF THE INVENTION

In one aspect the invention provides a nucleotide sequence encodingsubstantially the amino acid sequence shown in FIG. 10 (SEQ ID No: 2) ora functional equivalent of said nucleotide sequence.

The term functional equivalent is used in this context to encompassthose sequences which differ in their nucleotide composition to thatshown in FIG. 10 (SEQ ID No: 1) but which, by virtue of the degeneracyof the genetic code, encode polypeptides having identical orsubstantially identical amino acid sequences. It is intended that theterm should generally apply to sequences which are sufficientlyhomologous to the sequence of the invention that they can hybridise tothe complement thereof under stringent hybridisation conditions (eg asdescribed by Sambrook et al 1989, ie washing with 0.1×SSC, 0.5% SDS at68° C.); such equivalents will preferably possess at least 86%, morepreferably at least 90%, and most preferably at least 95%, sequencehomology (ie sequence similarity) with the sequence of the invention.Sequence homology is suitably determined using the ‘MEGALIGN’ program ofthe software package DNAStar (MEGALIGN and DNAStar are Trade Marks). Itwill be apparent to those skilled in the art that the nucleotidesequence of the invention may also find useful application when presentas an “antisense” sequence. Accordingly, functionally equivalentsequences will also include those sequences which can hybridise, understringent hybridisation conditions, to the sequence of the invention(rather than the complement thereof). Such “antisense” equivalents willpreferably possess at least 86%, more preferably at least 90%, and mostpreferably 95% sequence homology with the complement of the sequence ofthe invention.

In another aspect, the invention provides a nucleotide sequencecomprising substantially the sequence of B2 shown in FIG. 3 (SEQ ID No:3), or a functional equivalent thereof.

In a further aspect, the invention provides a nucleotide sequencecomprising substantially the sequence of B4 shown in FIG. 3 (SEQ ID No:4), or a functional equivalent thereof.

Another aspect of the invention provides a nucleotide sequencecomprising substantially the sequence of B10 shown in FIG. 3 (SEQ ID No:5), or a functional equivalent thereof.

Yet a further aspect of the invention provides a nucleotide sequencecomprising substantially the sequence of B1 shown in FIG. 3 (SEQ ID No:6), or a functional equivalent thereof.

In another aspect the invention provides a nucleotide sequence encodingsubstantially the amino acid sequence of B6 shown in FIG. 4 (SEQ ID No:7), or a functional equivalent thereof.

The term functional equivalent in this context has the same generalmeaning as discussed above, although equivalents for B2, B4, B10 and B6will preferably possess at least 90%, more preferably at least 95%,sequence homology with the relevant sequence of the invention, whileequivalents for B1 will preferably possess at least 97% sequencehomology with the sequence of the invention.

The sequences of the invention are part of novel wheat SBEII genes, withB1 being a novel subclass of the known class of SBEII genes, referred toherein as SBEII-2, with the novel subclass being called SBEII-2B. Theremaining sequences are all of a completely new class of wheat SBEIIgenes, referred to herein as SBEII-1. The sequences have been found tofall into 3 sub-classes, to be discussed below.

The novel wheat SBEII-1 genes that are the subject of this inventionhave strong sequence homology with the maize SBEIIb gene. The wheatSBEII-1 genes are thought to have similar functional properties to themaize SBEIIb gene. On this basis it is expected that by geneticmanipulation of the wheat SBEII-1 gene it will be possible to influenceproperties of starch produced by a plant, including the gelatinisationtemperature and rheological properties of starch, in a manner analogousto manipulation of the maize SBEIIb gene described in WO 97/22703. Thecontent of WO 97/22703 is incorporated herein by reference.

The present invention also includes within its scope a portion of any ofthe above sequences, comprising at least 500 base pairs and having atleast 90% sequence homology to the corresponding portion of the sequencefrom which it is derived.

Although the coding sequences of the novel wheat SBEII-1 genes havestrong sequence homology with the maize SBEIIb gene, there is muchgreater divergence in the 3′ untranslated parts of the sequences, with amaximum of 31.8% homology between the 3′ untranslated sequences of wheatSBEII-1 and maize SBEIIb as is apparent from FIG. 8.

In another aspect the invention thus provides a nucleotide sequencecomprising substantially the sequence shown in FIG. 5 (SEQ ID No: 8),FIG. 6 (SEQ ID No: 9) or FIG. 7 (SEQ ID No: 10), or a functionalequivalent thereof.

The term functional equivalent in this context has the same generalmeaning as discussed above, but with equivalents preferably at least32%, more preferably at least 40%, 50%, 60%, 70%, 80% or 90% sequencehomology with the sequence of the relevant Figure.

It is thought such 3′ untranslated sequences may be useful, both insense and antisense function, in manipulation of starch properties byaffecting SBE expression in plants, as will be discussed below.

The sequence may include further nucleotides at the 5′ or 3′ end. Forexample, for ease of expression, the sequence desirably also comprisesan in-frame ATG start code, and may also encode a leader sequence.

The invention also covers a nucleic acid construct comprising anucleotide sequence or portion thereof in accordance with the inventionconveniently operably linked, in sense or antisense orientation, to apromoter sequence.

Also included within the scope of the invention is amino acid sequenceencoded by any of the nucleotide sequences of the invention.

The invention also provides vectors, particularly expression vectors,comprising the nucleotide sequence of the invention. The vector willtypically comprise a promoter and one or more regulatory signals of thetype well known to those skilled in the art. The invention also includesprovision of cells transformed (which term encompasses transduction andtransfection) with a vector comprising the nucleotide sequence of theinvention.

Nucleotide sequences in accordance with the invention may be introducedinto plants, particularly but not exclusively wheat plants, and it isexpected that this can be used to affect expression of SBE in the plantand hence affect the properties of starch produced by the plant. Inparticular, use of sequences in antisense orientation is expected toreduce or suppress enzyme expression. Additionally, it has recently beendemonstrated in other experimental systems that “sense suppression” canalso occur (i.e. expression of an introduced sequence operably linked inthe sense orientation can interfere, by some unknown mechanism, with theexpression of the native gene), as described by Matzke & Matzke 1995.Any one of the methods mentioned by Matzke & Matzke could, in theory, beused to affect the expression in a host of a homologous SBE gene.

It is believed that antisense methods are mainly operable by theproduction of antisense mRNA which hybridises to the sense mRNA,preventing its translation into functional polypeptide, possibly bycausing the hybrid RNA to be degraded (e.g. Sheehy et al., 1988; Van derKrol et al.,). Sense suppression also requires homology between theintroduced sequence and the target gene, but the exact mechanism isunclear. It is apparent however that, in relation to both antisense andsense suppression, neither a full length nucleotide sequence, nor a“native” sequence is essential. Preferably the “effective portion” usedin the method will comprise at least one third of the full lengthsequence, but by simply trial and error other fragments (smaller orlarger) may be found which are functional in altering thecharacteristics of the plant.

Thus, in a further aspect the invention provides a method of alteringthe characteristics of a plant, comprising introducing into the plant aneffective portion of the sequence of the invention operably linked to asuitable promoter active in the plant so as to affect expression of agene present in the plant. Conveniently the sequence will be linked inthe antisense orientation to the promoter. Preferably the plant is awheat plant. Conveniently, the characteristic altered relates to thestarch content and/or starch composition of the plant (i.e. amountand/or type of starch present in the plant). Preferably the method ofaltering the characteristics of the plant will also comprise theintroduction of one or more further sequences, in addition to aneffective portion of the sequence of the invention. The introducedsequence of the invention and the one or more further sequences (whichmay be sense or antisense sequences) may be operably linked to a singlepromoter (which would ensure both sequences were transcribed atessentially the same time), or may be operably linked to separatepromoters (which may be necessary for optimal expression). Whereseparate promoters are employed they may be identical to each other ordifferent. Suitable promoters are well known to those skilled in the artand include both constitutive and inducible types. Examples include theCaMV 35S promoter (e.g. single or tandem repeat) and the ubiquitinpromoter. Advantageously the promoter will be tissue-specific. Desirablythe promoter will cause expression of the operably linked sequence atsubstantial levels only in the tissue of the plant where starchsynthesis and/or starch storage mainly occurs.

The sequence of the invention, and the one or more further sequences ifdesired, can be introduced into the plant by any one of a number ofwell-known techniques (e.g. Agrobacterium-mediated transformation, or by“biolistic” methods). The sequences are likely to be most effective inaffecting SBE activity in wheat plants, but theoretically could beintroduced into any plant. Desirable examples include pea, tomato,maize, rice, barley, sweet potato and cassava plants. Preferably theplant will comprise a natural gene encoding an SBE molecule whichexhibits reasonable homology with the introduced nucleic acid sequenceof the invention.

In another aspect, the invention provides a plant cell, or a plant orthe progeny thereof, which has been altered by the method defined above.The progeny of the altered plant may be obtained, for example, byvegetative propagation, or by crossing the altered plant and reservingthe seed so obtained. The invention also covers parts of the alteredplant, such as storage organs. Conveniently, for example, the inventioncovers grain comprising altered starch, said grain being obtained froman altered plant or the progeny thereof. Grain obtained from alteredplants (or the progeny thereof) will be particularly useful materials incertain industrial applications and for the preparation and/orprocessing of foodstuffs and may be used, for example, in bakeryproducts.

In particular relation to wheat plants, the invention provides a wheatplant or part thereof which, in its wild type possesses an effectiveSBEII-1 gene, but which plant has been altered such that there is eitherreduced, increased or no effective expression of an SBEII-1 polypeptidewithin the cells of at least part of the plant. The plant may have beenaltered by the method defined above, or may have been selected byconventional breeding to be deleted for the SBEII-1 gene, the presenceor absence of which can be readily determined by screening samples ofthe plants with a nucleic acid probe or antibody specific for the wheatgene or gene product respectively.

The invention also provides starch extracted from a plant altered by themethod defined above, or from the progeny of such a plant, the starchhaving altered properties compared to starch extracted from equivalent,but unaltered, plants. The invention further provides a method of makingaltered starch, comprising altering a plant by the method defined aboveand extracting therefrom starch having altered properties compared tostarch extracted from equivalent, but unaltered, plants. It is believedthat use of nucleotide sequences in accordance with the invention willenable the production of starches, particularly wheat starches, having awide variety of novel properties. For example, it may be anticipatedthat plants altered to give a reduction in SBEII activity will give riseto a starch with a relatively higher proportion of amylose and a lowerproportion of amylopectin compared with that from unaltered plants.

In particular the invention provides the following: a plant (especiallya wheat plant) altered by the method defined above, containing starchwhich, when extracted from the plant, has an elevated gelatinisationonset and/or peak temperature as measured by DSC, compared to starchextracted from a similar, but unaltered, plant; a plant (especially awheat plant) altered by the method defined above, containing starchwhich, when extracted from the plant, has a elevated gelatinisationonset temperature (conveniently elevated by at least 3° C., possibly byat least 7° C., by at least 12° C. or possibly even by 15 to 25° C.) asmeasured by DSC compared to starch extracted from a similar, butunaltered plant; a plant (especially a wheat plant) altered by themethod defined above, particularly to reduce expression of SBEII-1polypeptide, containing starch which, when extracted from a plant, has ahigher amylose:amylopectin ratio compared to starch extracted from asimilar, but unaltered plant.

The present invention particularly covers starch extracted from a plantaltered by the method of the invention, particularly starch having anincreased gelatinisation temperature. Such starch is useful, eg inbakery products, having particular benefits in certain situations, andthe invention also covers products, particularly bakery products, madefrom such starch. The invention also covers starch extracted from aplant altered by the method of the invention and having an increasedamylose:amylopectin ratio.

The invention will be further described, by way of illustration, in thefollowing Examples and with reference to the accompanying drawings, inwhich:

FIG. 1 is a graph of viscosity versus time, showing a viscoamylgraphprofile for wheat starch during and after cooking;

FIG. 2 shows alignment amino acid sequence data of C terminal portionsof various known starch branching enzymes (SEQ ID Nos: 12 to 25),obtained from the European Molecular Biology Laboratory (EMBL) database,and for a novel wheat SBEII-1 sequence of the invention (OsbeII-1ALL)(SEQ ID No: 11) from clone 5A1, with consensus residues highlighted;

FIG. 2 a is a residue weight table showing the percent similarity andpercent divergence of the sequences shown in FIG. 2;

FIG. 3 shows aligned DNA sequence data for various recombinant clones(B2, B4, B10, A2, B1, B11) (SEQ ID Nos: 3, 4, 5, 26, 6, 27 respectively)containing wheat starch branching enzyme genes, representing two SBEclasses, SBEII-1 and SBEII-2, each of which includes three subclasses A,B and C, with residues differing from the consensus (majority) (SEQ IDNo: 53) highlighted;

FIG. 3 a is a residue weight table showing the percent similarity andpercent divergence of the sequences shown in FIG. 3;

FIG. 4 is an alignment of predicted amino acid sequences for clones B6(wheat SBEII-1) (SEQ ID No: 7) and B11 (wheat SBEII-2) (SEQ ID No: 28)against the corresponding regions of the maize SBEIIa (SEQ ID No: 29)and SBEIIb (SEQ ID No: 30) amino acid sequences, with residues differingfrom those of maize SBEIIb highlighted;

FIG. 4 a is a residue weight table showing the percent similarity andpercent divergence of the sequences shown in FIG. 4;

FIG. 5 shows the 3′ untranslated DNA sequence of clone B2 (SEQ ID No: 8)(wheat SBEII-1, sub-class A);

FIG. 6 shows the 3′ untranslated DNA sequence of clone B10 (SEQ ID No:9) (wheat SBEII-1, sub-class B);

FIG. 7 shows the 3′ untranslated DNA sequence of clone B4 (SEQ ID No:10) (wheat SBEII-1, sub-class C);

FIG. 8 shows aligned DNA sequence data for the 3′ untranslated region ofclones B10 (SEQ ID No: 9), B2 (SEQ ID No: 8) and B4 (SEQ ID No: 10) andmaize SBEIIb (ZMSBE2b) (SEQ ID No: 31), with residues differing fromthose of the B10 sequence highlighted;

FIG. 8 a is a residue weight table showing the percent similarity andpercent divergence of the sequences shown in FIG. 8;

FIGS. 9 a and 9 b show hybridisation of clone B1 (SBEII-2) and clone B2(SBEII-1), respectively, to HindIII-digested genomic DNA of ChineseSpring wheat nullisomic-tetrasomic lines;

FIG. 10 shows the DNA (SEQ ID No: 1) and predicted amino acid sequence(SEQ ID No: 2) of part of SBEII-1 clone 5A1;

FIG. 11 shows aligned amino acid sequence data for the wheat SBEII-1sequence of the invention, from clone 5A1 (OsbeII-1ALL) (SEQ ID No: 11),wheat SBEI-D2 (SEQ ID No: 32) of Rahman et al 1997 (TASBEID2), wheatSBE1 of Rapellin et al 1997 (SEQ ID No: 33) (TASBEI) and wheat SBEII-2of Nair et al 1997 (SEQ ID No: 34) (wheat SBEII-2), with residuesexactly matching the consensus (majority) (SEQ ID No: 54) highlighted;

FIG. 11 a is a residue weight table showing the percent similarity andpercent divergence of the sequences shown in FIG. 11;

FIG. 12 illustrates northern blotting of wheat grains harvested atvarious different intervals after anthesis and probed with SBEII-1 andSBEII-2 fragments;

FIG. 13 is a restriction map of plasmid pWxGS+;

FIG. 13 a shows the sequence (SEQ ID No: 55) of the promoter(HindIII-BamH1 fragment) in pWxGS+;

FIG. 14 is a restriction map of plasmid psRWXGUS1;

FIG. 15 is a restriction map of plasmid pVTWXGUS2;

FIG. 16 is a restriction map of plasmid pPBI-97-2;

FIG. 17 is a restriction map of plasmid psR97-26A-;

FIG. 18 is a restriction map of plasmid psR97-29A-;

FIG. 19 is a restriction map of plasmid psR97-50A-;

FIG. 20 is a restriction map of plasmid psR97-53A-;

FIG. 21 is a restriction map of plasmid p97-2C;

FIG. 22 is a restriction map of plasmid p97-2CWT1;

FIG. 23 is a restriction map of plasmid psC98-1;

FIG. 24 is a restriction map of plasmid psC98-2;

FIG. 25 is a restriction map of plasmid pUNI;

FIG. 26 shows the DNA sequence of the NptII Sac1 fragment of pUNI (SEQID No: 35); and

FIG. 27 is a restriction map of plasmid pUSN99-1;

FIG. 28 is a restriction map of plasmid pUSN99-2;

FIG. 29 is a partial restriction map of the predicted sequence (SEQ IDNo: 52) of a cloned fragment of p97-U3;

FIG. 30 is a restriction map of plasmid pPB196-36;

FIG. 31 is a restriction map of plasmid p97-dUG1;

FIG. 32 is a restriction map of plasmid p97-2BdUN1;

FIG. 33 is a schematic illustration of a particle bombardment chamber(not to scale);

FIG. 34 shows histochemical localisation of Ubi-GUS expression in seed(panel A), stem (panel B), floral (panel C) and leaf tissues (panel D)of wheat transformed with plasmid pAHC25;

FIG. 35 is a Southern blot of 26 progeny plants of transformant BW119which had been transformed with pAHC25.

FIG. 36 shows histochemical localisation of waxy-GUS expression inendosperm tissue of two independent transgenic wheat lines (in panels Aand B) transformed with the plasmid pWxGS+; and

FIG. 37 is a Southern blot of genomic DNA of putative primarytransformants digested with Sac1 and probed with the 1 kb Sac1 SBEII-1probe.

EXAMPLES Amplification and Characterisation of Two Classes of SBEII cDNAClones

A PCR based cloning strategy was devised for isolating starch branchingenzymes from wheat using conserved domains within the known cloned genesequences. Starch branching enzymes have been cloned from a number ofplant species and FIG. 2 shows amino acid sequence data, obtained fromthe European Molecular Biology Laboratory (EMBL) nucleotide database forvarious known starch branching enzymes as follows: —

-   Wheat SBEII-2 for Triticum aestivum (SEQ ID No: 12)-   ZM SBE2a (maize) for Zea mays (SEQ ID No: 13)-   ZM SBE2b (maize) for Zea mays (SEQ ID No: 14)-   Barley SBEIIa (SEQ ID No: 15)-   Barley SBEIIb (SEQ ID No: 16)-   RICBCE3 (rice SBEII type enzyme) for Oryza sativa (SEQ ID No: 17)-   RICESBE-1/97 (as above, including transit peptide sequence) (SEQ ID    No: 18)-   PSSBEIGEN (pea SBEI, which is in fact an SBEII-type sequence) for    Pisum sativum (SEQ ID No: 19)-   STSBE (potato SBEI type) for Solanum tuberosum (SEQ ID No: 20)-   TASBEI (wheat SBEI-2) for Triticum aestivum (SEQ ID No: 21)-   TASBEI D2 (SEQ ID No: 22)-   ZMSBEI (maize SBEI) for Zea mays (SEQ ID No: 23)-   RICBEI (rice SBEI) for Oryza sativa (SEQ ID No: 24)-   PSSBEIIGN (pea SBEII, which is in fact an SBEI-type sequence) for    Pisum sativum (SEQ ID No: 25)

FIG. 2 also shows sequence information for a novel wheat SBEII-1sequence of the invention, identified as OsbeII-1ALL (SEQ ID No: 11).

The alignment report of FIG. 2, and also FIGS. 3, 4, 8 and 11, wasprepared using Clustal method, with PAM 250 residue weight table foramino acid sequences and weighted residue weight table for DNAsequences. Sequence pair distances expressed as % similarity shown inFIGS. 2A and 3A, 4A, 8A and 11A are determined using a ‘MEGALIGN’program of DNAStar software, and correspond to sequence homologypercentages as specified above.

Alignment of the sequences shown in FIG. 2 reveals several domains whichare highly conserved. One such domain, MDKDMYD (SEQ ID No: 36), wasalmost completely conserved and it was assumed that this domain wouldalso be present in wheat starch branching enzyme genes. This motif waschosen as a target for an oligonucleotide sense primer (SBEA). 3′RACEPCR was carried out on endosperm first strand cDNA using the primers Roand SBE A.

Two populations of PCR products of approximately 1 kb and 1.2 Kb werecloned into the plasmid vector pT7Blue (Novagen). Plasmid DNA from 36putative recombinant clones was purified and the insert size estimatedby restriction analysis. Fifteen clones harbouring inserts of betweenapproximately 1 Kb and 1.2 Kb were selected for sequencing. Alignment ofthe sequence data obtained, using the MEGALIGN program of DNAStar,indicated that the 15 selected clones could be divided on the basis ofdegrees of homology into two different classes, which we have designatedSBEII-1 and SBEII-2. Furthermore, both the SBEII-1 and SBEII-2 classesmay each be further subdivided into three sub-classes, based on sequencedifferences (Table 1). It is thought the sub-division into threesub-classes probably arises because wheat comprises three homoeologousgenomes.

TABLE 1 Class Sub-Class Clone Number SBEII-1 A B2, B5, B6, B7, B12SBEII-1 B B10 SBEII-1 C A1, A13, B4 SBEII-2 A B11 SBEII-2 B B1, B9SBEII-2 C A2, C5

Comparison between sequences within either of the SBEII-1 or SBEII-2classes showed between 90 and 96.8% similarity. In contrast, sequencesimilarity between representatives of SBEII-1 and SBEII-2 classes onlydisplay between 58.8 and 60.0% homology in the region of comparison(FIGS. 3 and 3 a).

Furthermore, we have compared representative sequences from each SBEII-1and SBEII-2 class with the previously reported wheat SBEII clones, pWBE6(Mousley, 1994) and the very recently published SBEII (Nair et al.,1997). The results showed that each of the previously isolated SBEIIclones are highly homologous (>90%) to our SBEII-2 class (data notshown). Significantly, neither of the previously reported wheatsequences showed high homology to our SBEII-1 sequence. The isolationand characterisation of three forms of SBEII-1 (SBEII-1, sub-classes A,B & C) is novel. The SBEII-2 sub-class B is also novel, sub-classes Aand C corresponding to the sequences previously disclosed by Mousley(1994) and Nair et al (1997) respectively.

Alignment of the predicted amino acid sequences from representativeclones, B6 and B11 of the wheat SBEII-1 and SBEII-2 sequences(respectively) against the corresponding regions of the maize SBEIIa andSBEIIb amino acid sequences (FIGS. 4 and 4 a) indicate that the wheatSBEII-1 sequence (clone B6) is more similar to the maize SBEIIb sequence(88.7% similarity) than to the wheat SBEII-2 sequence and the maizeSBEIIa sequence (82.2% & 82.6% similarity respectively) and similarlythat the wheat SBEII-2 sequence is more similar to the maize SBEIIasequence (86.9% similarity) than to the wheat SBEII-1 and maize SBEIIbsequences (82.2% and 81.7% similarity respectively). We thus hypothesisethat the wheat SBEII-1 is phylogenetically more related to the maizeSBEIIb and that the wheat SBEII-2 is phylogenetically related to themaize SBEIIa sequences and that the corresponding wheat and maizesequences are likely to exhibit similar functional properties.

While the coding sequences of clones B2, B10 and B4 have strong sequencehomology to the maize SBEIIb gene, there is much greater divergence inthe 3′ untranslated parts of the sequences. FIGS. 5, 6 and 7 show the 3′untranslated sequences of clones B2, B10 and B4, respectively, and FIG.8 compares these sequences with the corresponding sequence of maizeSBEIIb.

Considering matters in more detail, experimental details were asfollows.

Plant Material

Triticum aestivum cultivar Rialto was grown in a glass house undersupplementary lighting and temperature control to maintain a 16 hourday-length at 18+/−1° C.

Recombinant DNA Manipulations and Sequencing

Standard procedures were performed essentially according to Sambrook etal., (1989). DNA sequencing was performed on an ABI automated sequencerand sequences analysed using DNASTAR software for Macintosh.

RNA Isolation for cDNA Cloning

RNA was extracted from Triticum aestivum cultivar Rialto endosperm,using a Purescript RNA isolation kit (Flowgen) essentially according tothe manufacturers recommendations. Briefly, endosperm tissue was frozenin liquid nitrogen and ground, for 2 min, to a fine powder using adismembrenator (Braun Biotech International). The ground tissue wasstored in liquid nitrogen prior to extraction. Approx. 100 mg of groundtissue was transferred to a 1.5 ml microcentrifuge tube and 1.2 ml of‘Lysis buffer’ was added to the tissue before mixing by inversion andplacing on ice for 10 minutes. Protein and DNA were precipitated fromthe cell lysate by adding 0.4 ml of ‘Protein-DNA Precipitation Solution’and mixing by inversion before centrifuging at 13,000×g at 4° C. for 20minutes. The supernatant was divided between two fresh 1.5 ml tubes eachcontaining 600 μl of iso-propanol. The RNA precipitate was pelleted bycentrifugation at 13,000×g at 4° C. for 10 minutes, the supernatant wasdiscarded and the pellets washed with 70% ethanol by inverting the tubeseveral times. The ethanol was discarded and the pellet air dried for15-20 minutes before the RNA was resuspended in 7.5 ml of ‘RNA HydrationSolution’.

Preparation of Wheat Endosperm cDNA Pool

Wheat endosperm cDNA pool was prepared from total RNA, extracted asdescribed above, using Superscript™ reverse transcriptase (LifeTechnologies) essentially according to manufacturers instructions.Briefly, five microgrammes of RNA, 10 pMol RoRidT17[AAGGATCCGTCGACATCGATAATACGACTCACTATAGGGA(T17)] (SEQ ID No: 37) andsterile distilled water to a reaction volume of 12 μl, in a 500 μlmicrocentrifuge tube, was heated to 70° C. for 10 minutes before beingquick chilled on ice. The contents of the tube were collected by briefcentrifugation before adding 4 μl 5× First Strand Buffer, 2 μl 0.1M DTTand 1 μl 10 mM dNTPs and, after mixing, incubating at 42° C. for 2 min.1 μl of Superscript™ was added and, after mixing, incubation continuedfor 1 hour. The reaction was inactivated by heating to 70° C. for 15min. 150 μl of T₁₀E₁ was added to the reaction mix and the resultingcDNA pool was used as a template for amplification in PCR.

PCR Amplification of SBEII Sequences from Endosperm cDNA Pool

SBEII sequences were amplified from the endosperm cDNA pool usingprimers Ro [AAGGATCCGTCGACATC] (SEQ ID No: 38), which is complementaryto the Ro region of the RoRidT17 primer used to synthesise the cDNApool, and the SBEII specific primer, SBEA [ATGGACAAGGATATGTATGA] (SEQ IDNo: 39). SBEA was designed to be homologous to the MDKDMYD (SEQ ID No:36) motif which is situated approx. 1 kb from the 3′end of the maturepeptide coding sequence. PCR was carried out in a 50 μl reaction,comprising 5 μl of the cDNA pool, 25 pmol Ro, 50 pmol SBEA, 5 μl 5×Taqbuffer, 4 μl 25 mM Mg²⁺, 0.5 μl 20 mM dNTPs, and 1.25 u Taq polymerase.All of the reaction components were mixed, except for the Taqpolymerase, before being pre-heated to 94° C. for 7 min and then cooledto 75° C. for 5 min. Whilst the reaction mixtures were held at 75° C.the Taq polymerase was added and, after mixing well, the reactions werethermocycled at (94° C.-30 sec, 50° C.-30 sec, 72° C.-1 min)×30 cycles,followed by a final 10 min extension step at 72° C.

PCR products were purified by phenol/chloroform and chloroformextraction before ligation with pT7 Blue (Novagen) according tomanufacturers recommendations. Putative SBE clones were initiallycharacterised by standard plasmid DNA purification methods andrestriction digestion. Representative clones harbouring a range ofdifferent sized inserts were selected for sequencing.

Chromosomal Location of SBE Genes in Wheat

The Chinese Spring wheat nullisomic-tetrasomic lines as described inSears (1966) were used for assignment of the SBE sequences chromosomelocations. Ditelosomic lines (Sears, 1966) were used to determine thechromosome arm location. The Betzes barley ditelosomic addition lines inwheat are described in Islam (1983).

The chromosomal location of the two families of SBEII sequences(SBEII-1, SBEII-2) was determined by probing wheat nulli-tetra andditelosomic stock lines with gel-purified inserts of the various clones.FIG. 9 a shows the hybridisation obtained with an SBEII-2 (clone B1)probe on HindIII digested DNA. The euploid Chinese Spring gives 3 bands,one of which is missing in turn in the lines nullisomic for chromosomes2A, 2B and 2D. The same blot was re-probed with a SBEII-1 specific probe(clone B2). This yields an entirely different hybridisation profile(FIG. 9 b), demonstrating the specificity of the probe used. Again bandsare missing in each of the lines nullisomic for 2A, 2B and 2D. the samebanding pattern was observed using the SBEII-1 clones B2 and B4. Thusthe SBEII sub-family 1 and 2 gene sequences lie on the wheat group 2 setof homeologous chromosomes.

Ditelosomic addition lines were used to identify the arm location ofthese genes (data not shown). This revealed that the SBEII-1 and SBEII-2sequences are both located on the long arms of the homeologous group 2chromosomes of wheat.

Barley addition lines were used to determine whether homologoussequences are present in barley. These showed that sequences homologousto the wheat SBEII-1 and SBEII-2 sequences are located on the long armsof barley chromosome 2H.

RNA Isolation and Northern Blotting

Wheat grains were harvested at appropriate intervals and frozen inliquid Nitrogen before grinding to a fine powder using either a BraunMikrodismembrator™ or a pestle and mortar. Total RNA was isolated usingthe RNAqueous™ (Ambion Inc) Kit according to the manufacturersinstructions, or with the following method. Frozen powdered grain wasmixed with a 10× volume of 0.2M Tris-HCl pH9, 0.4M NaCl, 25 mM EDTA, 1%SDS, 1% PVPP, 0.25% Antifoam A, and 0.1M DTT. This mixture was extractedtwice with an equal volume of phenol/chloroform/isoamyl alcohol(25:24:1), the nucleic acids precipitated from the aqueous phase by theaddition of 0.8 volumes of isopropanol, and the resulting pelletdissolved in H₂O. The RNA was then selectively precipitated by theaddition of 1 volume of 4M LiCl, incubated at 4° C. overnight, and theresulting pellet dissolved in sterile distilled H₂O. 15 μg of total RNAwas electrophoresed on a 1% agarose, 2.21M Formaldehyde, 40 mM MOPSpH7.0, 10 mM sodium acetate, 1 mM EDTA gel, in a 40 mM MOPS pH7, 10 mMsodium acetate, 1 mM EDTA running buffer at 1 V/cm overnight. Gels wereplaced in a 50 ng/ml solution of Ethidium Bromide in water for 30minutes, de-stained in water for 2 hours, and visualised and photographsunder UV light. The gels were then washed briefly in sterile distilledH₂O, then blotted onto HyBond N⁺™ (Amersham International), according tostandard protocols (Sambrook et al, 1989) overnight. Blots were thendismantled and air-dried before UV fixing at 312 nm for 2 minutes.

Probe Isolation and Purification

5-10 μg of the plasmids pUN1 and psR98-29 were digested with Sst1 (LifeTechnologies Ltd) according to the manufacturers instructions, torelease fragments of approximately 0.8 kb (NptII) and 1 kb (SBEII-1)respectively. 5-10 μg of the plasmid pVT96-54 was digested with BamH1 torelease a SBEII-2 fragment of approximately 1.2 kb. Digests wereelectrophoresed on 1% low melting point agarose gels. The gene specificfragments were excised and the DNA purified using a Wizard™ GelPurification Kit (Promega).

Probe Labelling, and Hybridization

25 ng of the appropriate probe (Maize Waxy promoter, NptII, WheatSBEII-1 or Wheat SBEII-2 fragments) were radiolabelled using theRediprime 11™ system (Amersham International) using α³²PdCTP (AmershamInternational) according to manufacturers instructions. Blots werehybridized overnight at 65° C. in 0.6M NaCl, 20 mM Pipes, 4 mMNa₂EDTA2H₂O, 0.2% gelatin, 0.2% Ficoll 400, 0.2% PVP-360, 10 mMNa₄P₂O₇10H₂O, 0.8% SDS, 0.5 mg/ml denatured salmon sperm DNA. Posthybridization washes were carried out in 30 mM NaCl, 2 Mm NaH₂PO₄.2H₂O,0.2 mM Na₂EDTA.2H₂O, 0.1% SDS at room temperature for 7 minutes, then65° C. for 10 minutes. Filters were exposed to Kodak BioMax MR™(Amersham International) film at −70° C. Blots were stripped by washingin 15 mM NaCl, 1 mM NaH₂PO₄.2H₂O, 0.1 mM EDTA at 90° C. for 10 minutes,or until no counts above background remained.

Extension of the SBEII-1 3′ Sequence Towards the 5′ End of the MaturePeptide

We have exploited the sequence divergence between our wheat SBEII-1 andSBEII-2 sequences to design the SBEII-1 specific 3′ primer, Sb4. Thisprimer was used in conjunction with an SBEII specific 5′ primer toextend the novel SBEII-1 sequence using a PCR-based approach.

To extend the SBEII-1 3′ sequence towards the 5′ end of the maturepeptide, a second conserved domain was identified and an oligonucleotidesense primer, AGSBEI, designed. PCR amplification from the endospermfirst strand cDNA pool was carried out using the AGSBEI-Sb4 primer pair.Separation of the amplification products by electrophoresis through a 1%(w/v) agarose gel (data not shown) showed that the reaction yielded adistinct band of approx. 2.2 kb. The approx 2.2 kb amplificationproducts were excised from the gel, ligated with PT7Blue and transformedinto competent Novablue E. coli cells. Following overnight culture, nineputative recombinant clones were selected for further analysis.Screening of each of the selected clones using vector specific primersindicated that clones 5A1, 5A2, 5A5 and 5A9 harboured inserts of thepredicted size. Of these clone 5A1 (which falls in sub-class C) wasselected for sequencing (FIG. 10). The amino acid sequence of FIG. 10corresponds to the OsbeII-1ALL sequence of FIG. 2. Although not fulllength the predicted open reading frame includes nucleotides 44 throughto 1823 and encodes a 593 amino acid peptide. Based on similarities withthe maize genes, it is estimated that this sequence is missingapproximately 230 amino acids out of a predicted total of approximately830 amino acids. On this basis, the partial sequence represents about70% of the coding sequence. Multiple sequence alignment of this SBEII-1sequence with recently published wheat SBEII-2 (Nair et al., 1997), SBEI(Rapellin et al., 1997) and SBEI-D2 (Rahman et al., 1997) sequencesshowed that the SBEII-1 sequence has similarity indices of 69.6%, 31.2%and 46.7% to SBEII-2, SBEI and SBEI-D2 respectively (FIGS. 11 and 11 a).This demonstrates that the SBEII-1 sequence differs from the publishedwheat SBE sequences, and confirms the analysis of the 3′ sequencealignment (FIG. 3). The increase in relative homology when compared tothe values obtained following 3′ sequence alignment results from thefact that the central domain of SBEs is highly conserved (Burton et al.,1995; Gao et al., 1997). However, it is clear that this cloned wheatSBEII-1 sequence is significantly different from previously publishedwheat SBE sequences and represents a novel sequence.

Full experimental details were as follows.

SBEII-1 sequences were extended toward the 5′ end of the mature peptideby amplification from the endosperm cDNA pool using the SBEII-1 specificprimer Sb4 [TTTTCTTCACAACGCCCTGGG] (SEQ ID No: 40) in conjunction withthe primer AGSBEI [TGTTTGGGAGATCTTCCTCCC] (SEQ ID No: 41). AGSBEI wasdesigned to be homologous to the GVWEIFLP (SEQ ID No: 42) motif which isconserved in all known SBE sequences and is situated toward the 5′ endof the mature peptide coding sequence. PCR was carried out in a 50 μlreaction, comprising 5 μl of the cDNA pool, 50 pmol Sb4, 50 pmol SBEA1,5 μl 5×Taq buffer, 4 μl 25 mM Mg²⁺, 0.5 μl 20 mM dNTPs, and 1.25 u Taqpolymerase. All of the reaction components were mixed, beforethermocycling at (94° C.-45 sec, 55° C.-30 sec, 72° C.-1 min 30sec)×30cycles, followed by a final 10 min extension step at 72° C.Amplification products were separated by electrophoresis through a 1%(w/v) agarose gel and specific amplification products of the predictedsize were excised from the gel. The DNA was eluted from the gel sliceusing QIAGEN's gel extraction kit according to the manufacturersrecommendations before ligation with pT7 Blue (Novagen). Ligation wascarried out in a 10 μl reaction volume comprising 7.5 μl purifiedamplification product, 1 μl 10× ligation buffer, 1 μl pT7Blue and 0.5 μlT4 DNA ligase (Amersham). The reaction components were mixed well beforebeing placed at 4° C. overnight. Following overnight incubation, half ofthe ligation reaction was used to transform competent Novablue E. colicells (Novagen). Transformed cells were plated out onto LB platessupplemented with X-gal (40 μgml⁻¹), IPTG (0.1 mM), Carbenicillin (100μgml⁻¹), and Tetracycline (12.5 μgml⁻¹), before placing at 37° C.overnight. Putative recombinant clones were initially screened for thepresence of an insert by colony PCR using the vector specific primersT7B and U19. Insert positive clones were then screened using an insertspecific primer in conjunction with either T7B or U19 primers todetermine the orientation of the insert within the multiple cloning siteprior to sequencing.

Southern Blot Analysis

Southern analyses of the pre-made nulli-tetra and ditelosomic blots werecarried out essentially as described in Jack et al (1994).

The SBEII-1 clones discussed above have been cloned into transformationvectors for transformation of wheat.

Northern Blot Analysis

Northern blots were prepared from total RNA from developing wheat grainsof the cultivar Bobwhite. FIG. 12 shows a northern blot of RNA fromwheat grains of the cultivar Bobwhite grown in the glasshouse asdescribed and harvested between 5 and 29 days after anthesis. The blotwas probed with the 1 kb Sac1 SBEII-1 fragment and subsequently(following blot stripping) with the 1.2 kb BamH1 SBEII-2 fragment, bothfragments purified and labelled as described. In FIG. 12 panel A showsthe Ethidium Bromide-stained RNA gel prior to northern transfer. Panel Bshows the results of probing with the SBEII-1 probe and panel C showsthe results of probing with the SBEII-2 probe. Comparing within andbetween panels B and C differences can be observed in the relativeintensities of the signals at the different time points. In particular arelatively stronger signal intensity is observed with the SBEII-2 probefor the 5 day time point than with the SBEII-1 probe, indicating thatthe transcript profiles for SBEII-1 and SBEII-2 are distinct, suggestingthat the two gene families (SBEII-1 and SBEII-2) are differentiallyexpressed during grain development. The size of the transcripts observedfor both SBEII-1 and SBEII-2 is approximately 3.5 kb. However theSBEII-2 transcript is slightly smaller than the SBEII-1 transcript.

Plasmid Constructions

Standard molecular biology procedures (Sambrook et al, 1989) were usedfor plasmid constructions.

pWxGS+ (FIG. 13) comprising a maize granule bound starch synthase gene(Shure et al 1983) promoter-GUS-Nos fusion was obtained as a gift toUnilever Research from Sue Wessler (University of Georgia, Athens, USA)and may be obtained on request from that source. The promoter in pWxGS+is approximately 1.5 kb in length and represents a truncated version ofa similar, but larger promoter fragment described in Russell & Fromm(1997). The sequence of the promoter (HindIII-BamH1 fragment) in pWxGS+is presented in FIG. 13A (SEQ ID No: 55).

psRWXGUS1 (FIG. 14) was produced by inserting a Sac 1 linker[d(pCGAGCTCG)0] (New England Biolabs [NEB]) (NEB catalogue No 1044) intothe Sma1 site in pWxGS+.

pVTWXGUS2 (FIG. 15) was produced by inserting a BamH1 linker[d(pCGGGATCCCG)] (SEQ ID No: 43) (NEB catalogue No. 1071) into theEcl136II (an isoschizomer of Sac1 which gives blunt ends) site of pWxGS+

A Sac1 linker was inserted at the XbaI site (which had been bluntedusing Klenow+dNTps) of the SBEII-1 Clone B6 in the plasmid pT7Blue toproduce an intermediate clone. The SBE sequence was then purified fromthis intermediate clone as a Sac1 fragment and ligated into the Sac1sites of psRWXGUS1 replacing the GUS gene sequence to produce theplasmids psR96-26 and psR96-29 representing antisense and senseorientations of the SBEII-1 sequence downstream of the Waxy promoter,respectively.

A BamH1 linker was inserted at the XbaI site (which had been bluntedusing Klenow+dNTps) of the SBEII-2 Clone B11 in pT7Blue to produce anintermediate clone. The SBE sequence was then purified from thisintermediate as a BamH1 fragment and inserted into the BamH1 sites ofpVTWXGUS2, replacing the GUS gene sequence, to produce the plasmidspVT96-50 and pVT96-53 representing antisense and sense orientations,respectively, of the SBEII-2 sequence downstream of the Waxy promoter.

pVT96-54. A BamH1 linker was inserted at the Xba1 site (which had beenblunted using Klenow+dNTPs) of the SBEII-2 clone B9 (equivalent to cloneB1) in pT7Blue to produce an intermediate clone. The SBEII-2 sequencewas then purified from this intermediate clone as a BamH1 fragment andinserted into the BamH1 sites of pVTWXGUS2, replacing the GUS genesequence, to produce the plasmid pVT96-54.

The Waxy-SBE-NOS sequences in the plasmids psR96-26 and psR96-29 andpVT96-50 and pVT96-53 were purified as HindIII/EcoRI fragments andinserted into the EcoRI/HindIII sites of plasmid pPBI-97-2 (also knownas p97-2) (FIG. 16). Plasmid pPBI-97-2 is described in European PatentApplication No. 97305694.8 (published as WO 99/06570). Following removalof the ampicillin resistance marker gene the resulting plasmids weredesignated psR97-26A- (clone B6 (SBEII-1, sub-class A) in antisenseorientation), psR97-29A- (clone B6 in sense orientation), and psR97-50A-(clone B11 (SBEII-2, sub-class A) in antisense orientation) andpsR97-53A- (clone B11 in sense orientation) as illustrated in FIGS. 17,18, 19 and 20, respectively.

p97-2C (FIG. 21) was produced by digesting the polylinker sites Ecl136II to SmaI in the plasmid pPBI97-2 (FIG. 16), ligating and selectingrecombinants in which the polylinker region from SmaI to Ecl136 II hadreinserted in the opposite orientation.

The Waxy-NOS sequences in psRWXGUS1 were transferred as a HindIII/EcoRIfragment into the HindIII/EcoRI sites of plasmid p97-2C to produce theplasmid p97-2CWT1 (FIG. 22).

pSC98-1 and pSC98-2. The 5′ extended SBEII-1 clone 5A1 in pT7Blue(comprising SBE sequence from coordinate 43 to 2003 bp in FIG. 10) wasdigested with EcoRI and Xbal, followed by ‘in-fill’ of overhangs usingKlenow polymerase and dNTPs. The resulting blunt ended SBE fragment wasgel purified and ligated to p97-2CWT1 (FIG. 22) which had been digestedwith Ecl136II and dephosphorylated using calf intestinal phosphatase.The resulting recombinants were screened by restriction digest analysisand clones comprising both orientations of the SBE sequence (withrespect to the waxy promoter) were identified. pSC98-1 (FIG. 23) is anantisense version and pSC98-2 (FIG. 24) is a sense version. Followingremoval of the ampicillin marker gene the resulting plasmids weredesignated pSC98-1A- and pSC98-2A- respectively.

Ubiquitin Promoter—NptII Selection Construct:pUN1

pUN1 was made in the following way:

A SacI linker was inserted at the SmaI site of the plasmid pAHC25(Christensen and Quail 1996) to produce an intermediate plasmid. The GUSgene was removed from this intermediate plasmid by digesting with SacIfollowed by self ligation and identification of recombinant moleculeslacking the GUS sequence to produce the plasmid pPBI95-9. pPBI95-9 wasdigested with EcoRI and following self ligation recombinant moleculeslacking the Ubi-BAR sequences were identified. The resulting plasmid isdesignated pPBI96-23. An NptII sequence was amplified as a PCR productusing the primers AG95-7: 5′GATGAGCTCCGTTTCGCATGATTGAACAAGATGG (SEQ IDNo: 44) and AG95-8: 5′GTCGAGCTCAGAAGAACTCGTCAAGAAGGC (SEQ ID No: 45),using pPBIBAG3 (Goldsbrough et al 1994 as template for the NptIIsequence. The amplified product was cloned into the SstI site ofpBluescript (Stratagene) and sequenced. The sequencing revealed that theNptII sequence was of the ‘mutant’ form rather than the wild-type as hadbeen expected. The ‘mutant’ form carries a single base change which isflanked by unique Nco1 and Sph1 sites. The pBluescript clone wasdigested with Nco1 and Sph1 to remove the region containing the singlebase change. Two oligonucleotides, (Npt1:CCCGACGGCGAGGATCTCGTCGTGACC(SEQ ID No: 46) and Npt2: CATGGGTCACGACGAGATCCTCGCCGTCGGGCATG) (SEQ IDNo: 47) were then annealed to each other to form an Nco1/Sph1 fragment.This was cloned into the Nco1/Sph1 digested Bluescript/Npt11 clone, andthe resulting clone was sequenced to confirm that the gene was now ofthe wild type form.

The NptII sequences was then purified as a Sac1 fragment and inserted atthe SacI site of pPBI96-23 to produce pUN1 (FIG. 25). pUN1 includes thewild-type ubiquitin promoter (Ubi promoter), which is also referred toas the ubiquitin regulatory system (abbreviated to URS). The orientationof the NptII sequence in pUN1 was determined by restriction digestanalysis. The sequence of the NptII Sac1 fragment is presented in FIG.26 (SEQ ID No: 35).

pUSN99-1 and pUSN99-2. The SBEII-1 (clone B6) sequence was purified as aSac1 fragment from the plasmid psR96-26 and inserted at the Sac1 site ofpPBI96-23 to produce the plasmids pUSN99-1 and pUSN99-2 (FIGS. 27 and28) representing sense and antisense orientations of the SBEII-1sequences respectively.

pPBI97-2BdUN1. pPBI92-2BdUN1 (also sometimes referred to as p97-2BdUN1)comprises a reconstituted ubiquitin regulatory system (referred tohereafter as a modified ubiquitin promoter or a modified ubiquitinregulatory system (mURS)) which lacks the two overlapping ‘consensusheatshock elements’ discussed in EP 0342926 and U.S. Pat. No. 5,614,399.The modified ubiquitin promoter was prepared via PCR amplification oftwo DNA fragments using maize genomic DNA as template, followed byligation of the two fragments to produce a single fragment lacking theconsensus heatshock (HS) elements. A Kpn1 restriction site wasengineered in place of the HS elements. The primers used were designedfrom sequence information published by Liu et al 1995 (EMBL DNA databaseaccession ZMU29159). To delete the HS elements and to replace with adiagnostic Kpn1 site the ubiquitin promoter and intron sequences wereamplified as two fragments using the primer combinations HS1+Ubi3-3 andHS2+Ubi5-2, the sequences of which are given below. Primers Ubi5-2 andUbi3-3 are homologous to sequences in the sequence published by Liu etal 1995. Primers HS1 and HS2 are homologous to sequences locatedimmediately 3′ and 5′ respectively of the two overlapping HS elements inthe ubiquitin promoter as described in EP 0342926 and U.S. Pat. No.5,361,399. Both of these primers have a Kpn1 tail at their 5′ ends.

Primers

HS1: (SEQ ID No: 48) 5-ATTAGGTACCGGACTTGCTCCGCTGTCGGC -3 HS2: (SEQ IDNo: 49) 5-TATAGGTACCGAGGCAGCGACAGAGATGCC -3 Ubi5-2: (SEQ ID No: 50)5-AGCTGAATCCGGCGGCATGGC -3 Ubi3-3: (SEQ ID No: 51)5-TGATAGTCTTGCCAGTCAGGG -3

The amplified products were subcloned into pGEM TEasy (Promega) toproduce the plasmids p97-U1 and p97-U2. The full-length (approx. 2 Kb)modified ubiquitin promoter was reconstructed by subcloning theKpn1-Sac1 fragment from p97-U1 into the Kpn1/Sac1 sites of p97-U2 toproduce p97-U3. A partial restriction map of the predicted sequence (SEQID No: 52) of the cloned fragment in p97-U3 is presented in FIG. 29.(The modified ubiquitin promotor (or mURS) is the subject of a copendingEuropean Patent Application filed by the present applicants on the sameday as the present application, under the reference C1235.01/M). Themodified ubiquitin promoter was transferred as a PstI fragment fromp97-U3 into plasmid pPBI96-36. The plasmid pBI96-36 (FIG. 30) comprisesthe GUS-Nos reporter gene fusion under the control of the wild-typeubiquitin promoter (derived from pAHC25) in a pUC plasmid backbone. Thepromoter replaces the wild-type ubiquitin regulatory system in pPBI96-36to produce an intermediary plasmid p97-dUG1 (FIG. 31).

Construction of pPBI97-2BdUN1

The Ubi-Nos sequences in pPBI96-23 were transferred as an EcoRI-HindIIIfragment into the EcoRI and HindIII sites of p97-2B (plasmid p97-2B isdescribed in European Patent Application No. 97305694.8 published as WO99/06570) to produce the plasmid p97-2BUbiNos. The modified ubiquitinpromoter was purified as a HindIII/SacI fragment from p97-dUG1 (FIG. 31)and transferred into the HindIII and SacI sites of p97-2BUbiNos,replacing the wild-type ubiquitin promoter to produce p97-2BdUbiNos. TheNptII sequence in pUN1 was purified as a SacI fragment and transferredinto the SacI site of p97-2BdUbiNos to produce pPBI97-2BdUN1 (FIG. 32).Following removal of the ampicillin resistance marker using the methodas described in WO 99/06570, the resulting plasmid as used for wheattransformation was designated p97-2BdUN1A.

pCaineo

pCaiNeo comprises the NptII gene under control of a CaMV35S promoter andmaize Adh1 intron. The plasmid is described in Fromm et al 1986.

Transformation of Wheat

The following plasmid combinations (co-bombardments) have been used inthe transformation of wheat plants:

TABLE 2 Plasmid combinations used in wheat transformation experiments.Starch gene construct/s Selection marker construct pAHC25 pWXGS+ pUN1pSR97-26A- antisense pUN1 or p97-2BdUN1 pSR97-29A- sense p97-2BdUN1 orpCaiNeo pSC98-1A- antisense p97-2BdUN1 pUSN-1 sense p97-2BdUN1 pUSN-2antisense p97-2BdUN1 pUSN-1 sense & pUSN-2 antisense pUN1 pSC98-2A-sense p97-2BdUN1

The wheat transformation methods used and described here are largelybased on those described by Barcelo and Lazzeri, 1995.

Embryo wheat plants of the spring cultivar Bobwhite and the wintercultivar Florida were grown in a glasshouse with 16 hr day lengthsupplemented with lights to maintain a minimum light intensity of 500umol m−²s−¹ at 0.5M above flag leaf. Glasshouse temperatures weremaintained at 19° C.+/−1° C. during the day and 14° C.+/−1° C. at night.

Immature embryos of wheat were harvested from developing grain. Theseeds were harvested and embryos were cultured at approximately 12 daysafter anthesis when the embryos were approximately 1 mm in length. Seedswere first rinsed in 70% ethanol for 5 minutes and then sterilised in a10% solution of Domestos bleach (Domestos is a Trade Mark) for 15minutes followed by 6 washes with sterile distilled water. Followingremoval of the embryonic axis the embryos were placed axis surface facedown on agargel (Sigma catalogue no. A-3301) solidified MM1 media. Thegeneral recipe for MM1 is given in Appendix 1, and the recipes for thevarious constituents in Appendix 2. The embryos were maintained indarkness for one to two days at 24° C.+/−1° C. prior to bombardment.

The plasmids pAHC25, pCAiNeo, pUN1 and p97-2BdUN1 were used to provideselection markers in the combinations with starch gene constructs asdetailed in Table 2. pAHC25 (Christensen and Quail 1996) contains achimeric Ubi-BAR gene which provides selection of transformants tophosphinothricin, the active ingredient in herbicides BASTA™ andBialophos (see Block, M. de. et al 1987). The plasmids pCAiNeo (Fromm etal., 1986), pUN1 and p97-2BdUN1 contain chimeric promoter-NptII genefusions and provide selection of transformants against a range ofaminoglycoside antibiotics including kanamycin, neomycin, geneticin andparomycin.

Particle bombardments was used to introduce plasmids into plant cells.The following method was used to precipitate plasmid DNA onto 0.6 μmgold particles (BIO-RAD catalogue number 165-2262): A total of 5 μg ofplasmid DNA was added to a 50 μl sonicated for one minute suspension ofgold particle (@10 mg/ml) in a 1.5 ml microfuge tube. Following a briefvortex for three seconds 50 μl of a 0.5M solution of calcium chlorideand 20 μl of a 0.05M solution of spermidine free base were added to theopposite sides of the microfuge tube lid. The tube contents were mixedtogether by closing the lid and tapping the calcium chloride andspermidine to the bottom of the tube. Following a vortex for threeseconds the suspension was centrifuged at 13,000 rpm for 5 seconds. Thesupernatant was then removed and the pellet resuspended in 150 μl ofabsolute ethanol. This requires scraping the gold particles off theinside of the tube using a pipette tip. Following a further three secondvortex, the sample was centrifuged again and the pellet resuspended in atotal volume of 85 μl in absolute ethanol. The particles were vortexedbriefly and sonicated for 5 seconds in a Camlab Trisonic T310 water bathsonicator to ensure fine dispersion. An aliquot of 5 μl of the DNAcoated gold particles were placed in the centre of a macrocarrier(BIO-RAD catalogue no. 115-2335) and allowed to dry for 30 mins.Particle bombardment was performed by using a Biolisitc™ PDS-1000/He(BIO-RAD Instruments, Hercules Calif.) chamber which is illustratedschematically in FIG. 33, using helium pressure of 650 and 900 psi(rupture discs: BIO-RAD catalogue numbers 165-2327 and 165-2328respectively).

Referring to FIG. 33, the illustrated vacuum chamber comprises a housing10, the inner side walls of which include a series of recesses 12 forreceiving shelves such as sample shelf 14 shown at the fourth level downfrom the top of the housing. A rupture disc 16 is supported in a Hepressure shock tube 18 near the top of the housing. A support 20,resting in the second set of recesses 12 down from the top of thehousing, carries unit 22 that includes a stopping screen and a number ofrings 24, with 11 rings below the support 20 and 3-4 rings above thesupport 20. Macrocarrier 26 is supported at the top of unit 22. Theapproximate distance from the rupture disc 16 to the macrocarrier 26 is25 mm, with the approximate distance from the macrocarrier 26 to thestopping screen being 7 mm, and the approximate distance from thestopping screen to the sample shelf 14 being 67 mm. The top of unit 22is about 21 mm from the bottom of the shock tube 18, and the bottom unit22 is about 31 mm from the top of sample shelf 14.

Immature embryos were bombarded between 1 and 2 days after culture. Forbombardment the immature embryos were grouped into a circular area ofapproximately 1 cm in diameter comprising 20-100 embryos, axis side facedown on the MM1 media. The Petri dish (not shown) containing the tissuewas placed in the chamber on shelf 14, on the fourth shelf level downfrom the top, as illustrated in FIG. 33. The air in the chamber was thenevacuated to a vacuum of 28.5 inches of Hg. The macrocarrier 26 wasaccelerated with a helium shock wave using rupture membranes that burstwhen the He pressure in the shock tube 18 reaches 650 or 900 psi. Within1 hour after bombardment the bombarded embryos were plated on MM1 mediaat 10 embryos per 9 cm petri dish and then maintained in constantdarkness at 24° C. for 2-3 weeks. During this period somatic embryogeniccallus was produced on the bombarded embryos.

After 2-3 weeks the embryos were transferred onto agar-solidifiedregeneration media, known as R media, and incubated under 16 hrdaylength at 24° C. The general recipe for R media is given inAppendix 1. Embryos were transferred on fresh plates at 2-3 weekintervals. The composition of the regeneration media varied depending onwhich selection regime was to be used. For transformants bombarded withthe BAR gene the 3 amino solution was omitted and PPT (phosphinothricin)at 1 mg/L, rising to 3 mg/L over a period of three 2-3 week transferswas used for selection. For selection of transformants using the NptIIgene three different regimes were used: 1) Geneticin (GIBCO-BRLcatalogue no. 10131-019) was incorporated (at 50 mg/L) immediately ontransfer to regeneration media and maintained at 50 mg/L on subsequenttransfers to regeneration media. 2) & 3) Embryos were first transferredto regeneration media without selection for 12 days and 2-3 weeks,respectively, and thereafter transferred on to media containingGeneticin at 50 mg/L. After 2-3 passages on regeneration mediaregenerating shoots were transferred to individual culture tubescontaining 15 ml of regeneration media at half salt strength withselection at 3 mg/L PPT or 35 mg/L geneticin depending on whether theBAR gene of NptII gene had been used in the original bombardments.Following root formation the regenerated plants were transferred to soiland the glasshouse.

Genomic DNA Isolation and Southern Analyses

Southern analyses of primary transformants and progeny material werecarried out as follows: Freeze dried leaf tissues were ground briefly ina Kontes™ pestle and mortar, and genomic DNA extracted as described inFulton et al, 1995. 5 μg of DNA were digested with an appropriaterestriction enzyme according to the manufacturers instructions, andelectrophoresed overnight on a 1% agarose gel, after which the gel wasthen photographed, washed and blotted onto Hybond N+™ (AmershamInternational) according to the method of Southern using standardprocedures (Sambrook et al 1989). Following blotting, the filters wereair dried, baked at 65° C. for 1-2 hours and UV fixed at 312 nm for 2minutes.

Probe preparation and labelling for the Southern analyses of transformedmaterial was carried out as described above.

GUS histochemistry was performed essentially as described in Jefferson(1987).

Evaluation of the Ubiquitin Promoter for Constitutive Expression ofAssociated Transgenes.

The plasmid pAHC25 (Christensen and Quail, 1996) was transformed intowheat as described in previous sections. Transformants were selected onthe basis of resistance to phosphinothricin. Southern blot analyses werecarried out on the primary transformants to confirm integration of theplasmid sequences (data not shown). GUS histochemical analyses were alsocarried out and demonstrated that the ubiquitin promoter is capable ofmediating high levels of GUS expression in a range of wheat tissues.FIGS. 34 A, B, C & D show histochemical localisation of GUS expressionin the seed, stem, floral and leaf tissues respectively. Southern blotand GUS histochemical analyses were also carried out on self progenyfrom primary transformants to confirm that the transformation systemused is capable of producing transgenic plants which stably transmit theintegrated plasmid sequences to progeny plants. FIG. 35 shows a Southernblot of 26 progeny plants of transformant BW119 which had beentransformed with pAHC25. In this example genomic DNA from the progenyplants was digested with the restriction enzyme Sac1 and the blot wasprobed with the GUS gene coding sequence. The Southern blot results aresuggestive of the presence of two independently segregating integrationloci, each comprising concatamers of pAHC25 plasmid sequences.

Evaluation of the Maize Waxy Promoter for Endosperm-Specific Expressionof Associated Transgenes.

The plasmids pWxGS+ and pUN1 were co-transformed into wheat as describedin previous sections. Transformants were selected on the basis ofresistance to geneticin. Southern blot analyses were carried out on theprimary transformants to confirm integration of the plasmid sequences(data not shown). Gus histochemical analyses were also carried out todetermine the expression profile mediated by the maize waxy promoter.The majority of the transformants that expressed GUS exhibitedexpression specifically in endosperm tissue, demonstrating thesuitability of this promoter for mediating endosperm expression ofassociated transgenes. FIGS. 36 A & B shows endosperm specificexpression of GUS in seeds from two independent transformants. We didnot observe GUS expression in pollen grains as was seen by Russell andFromm (1997), however the construct they used also incorporated themaize hsp 70 intron which may conceivably have influenced expressionboth quantitatively and qualitatively.

Transformation of Wheat with Starch Gene Constructs.

The various construct combinations detailed in Table 2 wereco-transformed into wheat using the procedures as described in previoussections. Transformants were selected on the basis of resistance togeneticin. The primary transformants were confirmed positive by Southernblot analysis. Blots were sequentially probed with an NptII codingsequence probe and a SBE coding region probe. FIG. 37 shows an exampleof a Southern blot which comprises 22 putative transformants which hadbeen co-bombarded with pSR97-29A- or psR97-26A- and pUN1 or p97-2BdUN1.Genomic DNAs on this blot had been digested with Sac1. The blot wasfirst probed with the NptII probe. Lanes marked with an asteriskcorrespond to transformants which give a positive signal with the NptIIprobe. The blot shown in FIG. 37 was probed with the SBEII-1 1 kb Sac1fragment. The Sac1 digest is expected to release a 1 kb SBEII-1hybridising band from both pSR97-29A- and pSR97-26A- plasmid sequences,and the intensity of this band will vary depending on the copy number ofinserted plasmid sequences. As can be seen in FIG. 37 several additionalSBEII-1 hybridising bands are also observed. Five of these bands arepresent in all lanes and result from hybridisation to endogenous wheatSBEII-1 sequences. The additional bands of varying size which areobserved in the majority of lanes which show the 1 kb hybridising bandmost likely result from integration events in which one or more copiesof the plasmid had been linearised within the 1 kb SBEII-1 sequenceprior to integration. In the example shown in FIG. 37, of the 20 NptIIpositive plants, 16 were found to be co-transformed with the SBEII-1sequences, representing a co-transformation efficiency of 80%.

Differential Scanning Calorimetry (DSC)

When heated, an aqueous suspension of starch in excess water undergoes aco-operative endothermic transition known as gelatinisation, asdiscussed above, entailing a melting of the starch crystallites.Differential scanning calorimetry (DSC) measures the amount of energy(heat) absorbed or released by a sample as it is heated, cooled or heldin a constant (isothermal) temperature. DSC has been widely used tostudy the gelatinisation and retrogradation of starch.

DSC analyses were carried out on single grains or pools of 5 grains fromprimary transformants generated through transformation using each of thegene construct combinations detailed in Table 2.

Two different sample preparation and DSC methodologies were used:

Method 1:

Individual seed samples were crushed and ground using a pestle andmortar. The resulting bran was then separated and samples weighed into50 μm aluminium DSC pans. Water, three times by weight, was added andthe sample pans sealed. Analyses were performed using a Perkin-ElmerDSC-7 Robotic™ system equipped with an Intercooler II™, for sub-ambientconditions. Samples were heated from 25° C. to 80° C. at a heating rateof 5° C. min⁻¹. Gelatinisation enthalpy, onset and peak and endtemperatures were recorded. The thermograms were analysed using thePerkin-Elmer software programs (Thermal Analysis Software 7).Gelatinisation enthalpy is expressed in Joules (J)/gram (g) of sample.

Method 2:

Pools of 5 seeds from a single primary transformant, or single seedsfrom primary transformants, were milled using a Cemotec 1090™ SampleMill. The milled sample was then passed through a 250 micron sieve toseparate the bran from endosperm. Approximately 5 mg of the sievedsamples was then accurately weighed into 50 μl aluminium DSC pans.Water, three times by weight, was added and the sample pans sealed.Analyses were performed using a Perkin-Elmer Pyris 1™ DSC equipped withautosampler and Intracooler IP. Samples were heated from 40° C. to 85°C. at a heating rate of 10° C. per minute. The thermograms were analysedusing the Perkin-Elmer software programs (Pyris Software for Windows v3.5). Gelatinisation enthalpy, onset and peak and end temperatures wererecorded.

Using method 1, DSC analyses were performed on individual mature grainsof primary transformants, transformed with the plasmid combinationspSR97-26A-/pUN1, pSR97-26A-/p97-2BdUN1 and pSR97-29A-/p97-2BdUN1. Dataobtained were compared to data from control material which had beentransformed with one of the NptII selectable marker plasmids, but didnot contain any of the ‘starch’ plasmids. Table 3 summarises the averageonset, peak, end and enthalpy values for the selected material. Themajority of samples showed similar values to the control material.However, as can be seen from Table 3 onset, peak and end temperatureswere higher for a number of the transgenic samples compared to thecontrol material. For example, transformant BW 326 exhibits a 6.7° C.,4.9° C. and 4.6° C. increase in onset, peak and end temperatures(respectively) compared to the control sample.

Using method 2 a further series of DSC analyses were carried out onpools of 5 grains from primary transformants, transformed with theplasmid combinations psC98-1A-/p97-2BdUN1, pUSN-1/p97-2BdUN1,pUSN-2/p97-2BdUN1 and pUSN-1/pUSN-2/pUNI. Data obtained were compared todata from control material which had been transformed with one of theNptII selectable marker plasmids, but did not contain any of the‘starch’ plasmids. Table 4 summaries the onset, peak, end and enthalpyvalues for the selected pooled samples. In many cases there is evidencethat the ‘starch’ transgenic material shows onset, peak and endtemperatures which are greater than those observed for the controlmaterial. For example, transformant BW727 exhibits a 9.8° C., 8.7° C.and 9.1° C. increase in onset, peak and end temperatures (respectively)compared to the BW control sample 3, and a 7.6° C., 6.8° C. and 7.8° C.increase in onset, peak and end temperatures (respectively) compared tothe BW control sample 2.

TABLE 3 Results of DSC analyses on single grains using method 1. Datashown are the averages of between 2 and 6 individual grain samples(T_(o), T_(p) and T_(f) are onset, peak and end temperaturesrespectively). Line T_(o) T_(p) T_(f) ΔH Plasmid combination Code (° C.)(° C.) (° C.) (J/g) BW control sample 1 55.2 59.7 66.5 4.66pSR97-26A-/pUN1 BW283 57.1 60.4 65.0 2.12 BW135 57.2 62.1 68.6 4.86BW324 57.8 62.1 69.1 5.33 BW325 58.4 61.8 68.7 3.90 BW326 61.9 64.6 71.12.46 BW348 60.7 63.4 69.7 3.76 pSR97-26A-/p97-2BdUN1 F227 57.4 61.4 67.32.65 pSR97-29A-/p97-2BdUN1 F310 62.1 63.7 69.2 6.75 F312 59.0 62.3 66.81.16 BW335 56.2 60.8 69.1 4.63 BW353 59.5 62.7 70.8 3.21 BW354 55.4 61.768.9 4.28 BW355 57.9 61.5 68.0 3.95 BW357 55.3 60.6 68.0 3.74 BW363 56.762.5 67.9 1.13 BW367 59.0 62.5 68.2 2.17 BW369 57.9 60.9 65.9 1.04 BW37053.7 59.4 67.5 6.00 BW375 57.2 61.5 70.0 4.14 BW376 54.0 58.1 68.0 3.39BW377 53.4 60.9 69.2 2.60 BW380 54.6 61.6 67.6 2.16 BW390 56.8 61.2 68.51.29 BW399 57.4 62.7 67.9 1.77 BW400 60.6 63.6 68.1 0.64 BW341 51.6 59.066.4 1.97

TABLE 4 Results of DSC analyses on pools of 5 grains using method 2.T_(o), T_(p) and T_(f) are onset, peak and end temperatures respectivelyLine T_(o) T_(p) T_(f) ΔH Plasmid combination Code (° C.) (° C.) (° C.)(J/g) F control sample 1 60.1 63.9 68.0 6.30 BW control sample 2 59.364.0 68.4 5.94 BW control sample 3 57.08 62.09 67.08 4.28pSC98-1A-/p97-2BdUN1 BW449 59.3 62.9 67.9 3.95 BW477 57.7 63.6 70.6 8.30F492 62.3 66.4 70.2 7.60 F494 63.6 67.3 71.0 5.73 BW511 59.6 63.8 67.20.98 BW518 60.2 64.9 69.2 3.57 BW519 58.4 63.6 68.5 4.13 BW527 58.7 63.769.0 6.38 BW549 59.9 64.8 69.3 4.48 BW550 60.2 64.6 68.9 5.06 BW552 60.862.9 67.9 3.74 BW553 59.5 63.9 67.5 3.60 BW555 61.0 66.1 68.2 5.43 BW55762.7 66.9 71.0 5.08 BW559 61.6 65.9 70.8 5.08 BW563 61.4 65.1 69.4 1.90BW564 59.4 64.5 73.2 7.08 BW576 61.8 65.6 69.3 2.65 BW587 61.3 65.4 69.45.36 BW614 63.9 67.9 71.8 5.83 BW618 61.3 65.6 69.7 3.54 BW583a 58.963.7 68.0 3.54 BW631 61.5 65.6 69.7 4.52 BW633 61.9 66.0 70.2 5.12BW634a 60.8 64.9 70.2 5.10 BW637a 62.8 67.2 72.0 5.16 BW639 61.8 65.168.9 2.15 BW640a 62.2 66.7 71.0 3.23 BW642 63.2 67.2 70.9 4.90 BW69862.9 67.0 70.9 4.48 BW700a 63.8 67.6 71.2 3.41 BE524a 59.4 64.3 68.94.05 pUSN-1/p97-2BdUN1 BW622 59.0 64.1 68.7 4.32 BW628 56.2 63.3 66.06.09 BW645 57.5 65.6 69.5 5.97 BW646 61.6 66.4 67.7 3.99 BW647 61.3 65.469.0 3.47 BW648 59.8 64.4 68.8 4.65 BW649 61.3 65.6 70.1 5.07 BW656 59.964.6 69.2 5.38 BW660 62.0 67.3 71.0 4.23 BW661 61.5 65.8 69.6 3.88 BW66461.1 66.1 70.8 4.81 BW665 61.6 66.5 69.4 5.25 BW667 63.0 67.1 70.8 3.91BW672 63.0 68.1 71.9 5.43 BW673A 63.1 67.7 71.6 4.83 BW675 62.1 66.471.3 10.97 BW676 59.8 67.3 71.2 4.21 BW678 63.0 66.3 69.3 1.20 BW68060.8 65.3 70.1 4.94 BW701 62.3 67.5 72.2 4.70 BW706 63.0 67.3 71.3 4.94BW707 60.9 65.8 70.0 4.77 BW708 61.7 65.5 68.8 6.11 BW726 62.6 67.5 71.35.44 BW755 60.8 65.8 70.6 5.18 BW702 61.9 67.0 71.0 4.44 BW756 62.3 66.169.7 4.83 pUSN-2/p97-2BdUN1 BW625 62.7 68.2 73.8 4.27 BW653 60.4 65.370.1 6.52 BW704 60.9 66.2 70.2 4.19 BW718 61.3 66.9 71.2 4.15 BW719 62.267.2 71.7 5.32 BW722 64.8 67.5 70.0 2.14 BW740 63.4 67.9 72.3 5.67 BW74162.6 66.9 70.5 5.30 BW742 64.6 67.9 72.0 6.66 BW752 62.3 66.3 70.0 4.63pUSN-1/pUSN-2/pUN1 BW685 62.6 65.5 69.0 2.60 BW686A 61.9 66.3 70.2 4.45BW714 63.0 67.6 71.3 3.53 BW727 66.9 70.8 76.2 5.19 BW728 62.0 66.3 70.45.70 BW731 63.3 67.9 73.0 4.90 BW732 63.5 66.8 70.8 4.11 BW748 62.1 67.471.9 5.38 BW794 62.8 67.5 71.8 5.17

APPENDIX 1 Volume of stock per litre of 2x Constituent concentratedmedia Recipe for 2x concentrated MM1 media Macrosalts MS (10X stock) 200ml Microsalts L (1000x stock) 2 ml FeNaEDTA MS (100x stock) 20 ml [Sigmacatalogue F-0518] Modified Vits MS (x1000) 1 ml 3 amino acid solution(25x stock) 40 ml myo inositol 0.2 g (Sigma catalogue number I-3011)sucrose 180 g AgNO₃ (20 mg/ml stock) 1 ml Added after filtersterilisation Picloram (1 m/ml stock) 4 ml Added after filtersterilisation Filter sterilise and add to an equal volume of moulten 2xagargel (10 g/L). Recipe for 2x concentrated R media Macrosalts L7 (10Xstock) 200 ml Microsalts L (1000x stock) 2 ml FeNaEDTA MS (100x stock)20 ml Vits/Inositol L2 (200x stock) 10 ml 3 amino acid solution (25xstock) 40 ml Maltose 60 g 2,4-D (1 mg/ml stock) 200 μl added afterfilter sterilisation Zeatin cis trans mixed isomers 2 ml (Melford labscatalogue no. Z-0917) (5 mg/ml stock) added after filter sterilisationFilter sterilise and add to an equal volume of moulten 2x agar (16g/litre)

APPENDIX 2 Recipes for constituents of MM1 and R media Microsalts L(1000x stock) per 100 ml MnSO₄•7H₂O 1.34 g H₃BO₃ 0.5 g ZnSO₄•7H₂O 0.75 gKI 75 mg Na₂MoO₄•2H₂O 25 mg CuSO₄•5H₂O 2.5 mg CoCl₂•6H₂O 2.5 mg Filtersterilise through a 22 μm membrane filter Store at 4° C. Macrosalts MS(10X stock) per litre NH₄NO₃ 16.5 g KNO₃ 19.0 g KH₂PO₄ 1.7 g MgSO₄•7H₂O3.7 g CaCl₂•2H₂O 4.4 g NB: Dissolve CaCl₂ before mixing with othercomponents NB: Make up KH₂PO₄ separately in sterile H₂0, and add last.Store solution at 4° C. after autoclaving Modified MS Vits (1000x stock)Per 100 ml Thiamine HCl 10 mg Pyridoxine HCl 50 mg Nicotinic acid 50 mgStore solution in 10 ml aliquots at −20° C. 3 amino acid solution (25xstock) Per litre L-Glutamine 18.75 g L-Proline 3.75 g L-Asparagine 2.5 gStore solution in 40 ml aliquots at −20° C. Macrosalts L7 (10x stock)per litre NH₄NO₃ 2.5 g KNO₃ 15.0 g KH₂PO₄ 2.0 g MgSO₄•7H₂O 3.5 gCaCl₂•2H₂O 4.5 g NB: Dissolve CaCl₂ before mixing with other componentsNB: Make up KH₂PO₄ separately in 50 ml H₂0 and add last Store solutionat 4° C. after autoclaving Vits/Inositol (200x stock) 200x Stock Per 100ml Inositol 4.0 g Thiamine HCl 0.2 g Pyridoxine HCl 0.02 g Nicotinicacid 0.02 g Ca-pantothenate 0.02 g Ascorbic acid 0.02 g Store solutionin 40 ml aliquots at −20° C.

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1. An isolated nucleotide sequence encoding the amino acid sequence ofSEQ ID NO:7.
 2. A nucleic acid construct comprising the nucleotidesequence of claim
 1. 3. The nucleic acid construct of claim 2, whereinthe nucleotide sequence is operably linked in sense or antisenseorientation to a promoter sequence.
 4. The nucleic acid construct ofclaim 3, wherein the nucleic acid construct is an expression vector. 5.A host cell comprising the nucleic acid construct of claim
 2. 6. Amethod of altering the starch characteristics of a wheat plant,comprising introducing into the genome of the wheat plant a nucleotidesequence encoding the amino acid sequence of SEQ ID NO:7, wherein thenucleotide sequence is operably linked to a suitable promoter that isactive in the wheat plant.
 7. The method of claim 6, wherein thenucleotide sequence is linked in the antisense orientation to thepromoter.
 8. The method of claim 6, wherein the altered starchcharacteristics relate to the starch content and/or starch compositionof the wheat plant.
 9. A wheat plant, or progeny of said wheat plant,altered by the method of claim 6 and each comprising the introducednucleotide sequence.
 10. A part or cell of a wheat plant according toclaim 9, each comprising the introduced nucleotide sequence.
 11. Thewheat plant part or cell of claim 10,wherein the part is a storageorgan.
 12. A wheat plant according to claim 9, or part or cell thereof,each comprising the introduced nucleotide sequence and each containingstarch having an elevated gelatinization onset and/or peak temperatureas measured by differential scanning calorimetry (DSC) compared tostarch from an unaltered wheat plant.
 13. A method of making alteredstarch, comprising altering a wheat plant by the method of claim 6 andextracting therefrom starch having altered properties compared to starchextracted from unaltered wheat plants.